CN118580253A - Near-infrared photosensitizer activated by microacidic environment of tumor and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of biomedical synthesis technology, and specifically discloses a near-infrared photosensitizer that can be activated by a tumor micro-acid environment, and a preparation method and application thereof. The near-infrared photosensitizer that can be activated by a tumor micro-acid environment has a chemical structure as shown in any one of formulas (I) to (VI). The near-infrared photosensitizer that can be activated by a tumor micro-acid environment uses a cyanine or semi-cyanine derivative with good biocompatibility and high near-infrared light absorption ability as a basic skeleton, introduces an ether oxygen bond as an acid response group at the ortho position of the indole nitrogen atom, and adjusts the ability of the photosensitizer molecule to produce active oxygen by replacing different halogen atoms with _R1 , and the _R2 group is a hydroxyl, amino or biomarker responsive group. The photosensitizer of the present invention is specifically activated in tumors, has strong near-infrared absorption and fluorescence emission capabilities, has a photodynamic therapy effect, can achieve precise treatment of tumor sites, reduce systemic toxicity, and provide a new strategy for precision medicine for tumors.

Description

Near-infrared photosensitizer activated by microacidic environment of tumor and preparation method and application thereof

Technical Field

The present invention relates to the field of biomedical synthesis technology, and in particular to a near-infrared photosensitizer that can be activated by a micro-acidic environment of a tumor, and a preparation method and application thereof.

Background Art

Cancer has a high incidence and mortality rate, which seriously endangers people's life and health. Photodynamic therapy (PDT) is an emerging tumor treatment program. It uses photosensitizers to transfer laser energy to dissolved oxygen in tumors, producing cytotoxic singlet oxygen, thereby achieving the purpose of killing tumor cells. This light-controlled therapy has the following advantages: (1) Very little tissue trauma: Through light treatment, it is non-invasive; with the help of optical fiber, endoscope and other interventional technologies, the laser can also be guided to deep inside the body for treatment, avoiding the trauma and pain caused by thoracotomy, laparotomy and other surgeries. (2) High spatiotemporal selectivity and low systemic toxicity: Only when the photosensitizer enters the tissue and reaches a certain concentration and is exposed to sufficient light will it trigger a photodynamic reaction and kill the targeted cells. The part that is not exposed to light does not produce this killing reaction, causing little damage to organs and tissues in other parts, and does not affect hematopoietic function. Therefore, this selective treatment method that only irradiates the diseased tissue locally has low systemic toxic side effects, which is difficult to achieve with many other treatment methods. (3) Good applicability: Different types of tumor cells have different sensitivities to radiotherapy and chemotherapy, so their application is limited. Photodynamic therapy is effective for lesions of different cell types and has a wide range of applications. (4) Repeatable treatment: Tumor cells have no resistance to photosensitive drugs, and patients will not experience increased toxicity due to multiple treatments, so repeated treatments are possible.

At present, the photosensitizers used in clinical practice are mainly porphyrin derivatives. These photosensitizers have the following main problems: (1) The diffuse distribution of photosensitizer drugs throughout the body causes excessive accumulation of drugs in normal tissues, causing clinical side effects such as skin phototoxicity that affect the patient's quality of life. For example, It is one of the most widely used photosensitizer drugs in clinical practice in recent years. Its main ingredient is a derivative of hematoporphyrin. The drug lacks tumor selectivity and is widely distributed in the skin throughout the body after administration. It causes severe skin inflammation under sunlight. The FDA requires that patients must not expose their skin and eyes to sunlight for up to 1 month after taking the drug, which seriously affects their lifestyle. (2) The laser penetration depth of photosensitizers is shallow, and the therapeutic effect on deep tumors is poor. (3) The active oxygen production rate of photosensitizers is generally low. In addition, the inherent hypoxic characteristics of the tumor microenvironment and the continuous consumption of oxygen during treatment greatly weaken the effect of photodynamic therapy. Therefore, higher doses of photosensitizer drugs have to be used to meet treatment needs, thereby increasing the long-term safety risks of medication. Therefore, overcoming these problems and developing new photosensitizer molecules are of great significance to the clinical application of photodynamic therapy.

Therefore, the prior art still needs to be improved and developed.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a near-infrared photosensitizer that can be activated by a micro-acidic environment of a tumor and a preparation method and application thereof, aiming to solve the problems of high skin phototoxicity, shallow treatment depth and low reactive oxygen production of existing photosensitizers.

The technical solution of the present invention is as follows:

In a first aspect of the present invention, a series of near-infrared photosensitizers that can be activated by a slightly acidic environment of a tumor are provided, having a chemical structure shown in any one of formula (I) to formula (VI):

Wherein, R ₁ is H, F, Cl, Br or I atom, R ₂ is -OH, -NH ₂ , A is a self-eliminating group, and B is a biomarker responsive group.

The photosensitizer provided by the present invention uses cyanine/hemicyanine as the parent structure, and introduces an ether oxygen bond as an acid response group at the ortho position of the indole nitrogen atom. The ability of the photosensitizer molecule to generate active oxygen is adjusted by replacing different halogen (fluorine, chlorine, bromine and iodine) atoms by _R1 . For the semi-cyanine skeleton, in addition to the acid response group, an _R2 group (hydroxyl, amino or a group with biomarker responsiveness) is connected to one end of the photosensitizer molecule. Among them, the cyanine molecular skeleton of formula (I) to formula (III) has a high molar absorption coefficient and near-infrared photosensitivity, and the positive charge of the indole structure makes it easy to target mitochondria. The semi-cyanine molecular skeleton of formula (IV) to formula (VI) is a variant of the cyanine molecule, and has photophysical and photochemical properties very similar to those of the cyanine molecule, but has higher photostability and structural modifiability. The -OH of the acid-responsive group forms a closed-ring structure with an ether oxygen bond at the ortho position of the indole nitrogen atom. The photosensitizer is in an unactivated state, has no absorption and fluorescence in the near-infrared region, and has no near-infrared photodynamic therapy effect; however, after being activated in the slightly acidic environment of the tumor, the ether oxygen bond formed by the acid-responsive group breaks, forming an open-ring structure, and the molecule restores the classic cyanine/hemicyanine structure, which can produce strong near-infrared absorption and fluorescence, and has near-infrared photodynamic therapy effect. Different halogen atoms substituted by R ₁ in the photosensitizer have different atomic effects in the molecular photosensitization process, thereby obtaining a higher active oxygen yield. The R ₂ biomarker response group introduced in the hemicyanine skeleton cooperates with the acid response group to obtain a dual-responsive photosensitizer molecule with higher tumor selectivity.

It should be noted that the chemical structure of the photosensitizer All of them are indole structures, and the three can be equivalently replaced with each other in the present invention. However, as the conjugation degree of the molecular skeleton increases successively, the absorption and emission wavelengths thereof also increase successively.

Optionally, the self-eliminating group A is

Optionally, the biomarker responsive group B is:

Optionally, after being activated in a slightly acidic environment, the photosensitizers represented by formula (I) to formula (VI) have structures represented by corresponding formula (I') to formula (VI'):

Optionally, the near-infrared photosensitizer activated by the micro-acidic environment of the tumor has the following structural formula:

The use of the near-infrared photosensitizer molecule activated by the microacidic environment of the tumor or its pharmaceutically acceptable enantiomer, diastereomer, tautomer in the preparation of photosensitizer molecules is also within the protection scope of the present invention.

The second aspect of the present invention provides a method for preparing the near-infrared photosensitizer that can be activated by the micro-acidic environment of tumors, comprising the steps of:

Step S1: Compound 1 represented by Formula 1 and compound 4 represented by Formula 4 undergo condensation reaction to obtain compound 6 represented by Formula 6;

or the compound 2 represented by formula 2 and the compound 4 represented by formula 4 undergo a condensation reaction to obtain the compound 7 represented by formula 7;

or the compound 3 shown in formula 3 and the compound 4 shown in formula 4 undergo a condensation reaction to obtain the compound 8 shown in formula 8;

or the compound 1 represented by formula 1 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 9 represented by formula 9;

or the compound 2 represented by formula 2 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain a compound 10 represented by formula 10;

or the compound 3 represented by formula 3 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 11 represented by formula 11;

Step S2: Compound 6 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by a tumor slightly acidic environment as shown in formula (I);

Or compound 7 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (II);

Or compound 8 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (III);

Or compound 9 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (IV);

Or compound 10 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor as shown in formula (V);

Or compound 11 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (VI).

Wherein, the structural formulas of compounds 1 to 11 are shown in the following formulas 1 to 11:

Optionally, the temperature of the condensation reaction in step S1 is 50-100° C., and the time of the condensation reaction is 2-12 hours.

Optionally, the solvent used in the condensation reaction in step S1 is one or more of acetic anhydride, anhydrous methanol, anhydrous ethanol and anhydrous N,N-dimethylformamide, and the optional activating agent is one or more of sodium acetate, potassium acetate, potassium carbonate and cesium carbonate.

Optionally, the alkaline conditions required for the ring-closing reaction in step S2 include one or more of potassium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, ammonia water or triethylamine.

Optionally, the solvent used in the ring-closing reaction in step S2 is one or more of dichloromethane, methanol, ethanol, chloroform and N,N-dimethylformamide.

Optionally, the temperature of the ring-closing reaction is 0 to 50° C., and the time of the ring-closing reaction is 2 to 12 hours.

Optionally, the compound 1 in step S1 is prepared by a substitution reaction between compound 12 and compound 13. The temperature of the substitution reaction is 50 to 150° C., the time of the substitution reaction is 24 to 72 hours, and the solvent used in the substitution reaction is one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene and o-dichlorobenzene.

The structural formulas of compound 12 and compound 13 are shown below:

Optionally, the compound 3 in step S1 is prepared by the following steps:

Compound 14 undergoes a sulfurization reaction to obtain compound 15;

The compound 15 undergoes iodination reaction to obtain compound 16;

The compound 16 undergoes a substitution reaction with the compound 17 to obtain the compound 18;

The compound 18 undergoes a substitution reaction with the compound 13 to obtain the compound 19;

The compound 19 undergoes a hydrolysis reaction to obtain the compound 3.

Wherein, the structural formulas of the compounds 13 to 19 are as follows:

Optionally, the reaction temperature involved in the preparation step of compound 3 is 50-150° C., the reaction time is 24-72 h, and the solvent used in the reaction can be one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene and o-dichlorobenzene.

The third aspect of the present invention provides a use of the near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor, for preparing a tumor diagnostic agent and/or a tumor therapeutic agent.

Optionally, the tumor diagnostic agent comprises a fluorescent imaging agent or a photoacoustic imaging agent.

Optionally, the tumor treatment agent includes a photodynamic therapy agent or an agent for combining photodynamic therapy with other treatment options.

Optionally, the dosage form of the medicament is capsule, tablet, oral preparation, injection, suppository, spray or ointment.

The near-infrared dye molecules activated by the micro-acid environment of the tumor provided by the present invention can be used as photosensitizers for photodynamic therapy, and are prepared into preparations for various modes of administration (intravenous injection, microneedle patch, intraperitoneal injection or spraying). After the near-infrared photosensitizer activated by the micro-acid environment of the tumor is intravenously injected, since the pH of the normal tissue microenvironment is 7.4, the acid response group of the photosensitizer molecule exists in a closed-loop form, and the molecule is not activated, so obvious near-infrared light absorption and emission will not be detected in normal tissues, and under near-infrared light irradiation, the photosensitizer will not produce reactive oxygen species, so it will not cause damage to normal tissues. When the photosensitizer molecule is in a micro-acid environment of the tumor (pH <7), the acid response group exists in an open-loop form, and the photosensitizer molecules shown in formula (I) to formula (III) are activated, producing strong near-infrared absorption and emission, so that fluorescence or photoacoustic imaging can be performed, and reactive oxygen species can be produced under near-infrared light irradiation, thereby killing tumor tissues. After being further activated by specific biomarkers expressed in large quantities in tumors, the photosensitizer molecules shown in formula (IV) to formula (VI) can produce near-infrared light absorption and emission, guide photodynamic therapy through fluorescence or photoacoustic imaging, achieve precise visualization of photodynamic therapy, improve the treatment effect, and provide new ideas for the development of photodynamic therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the activation principle of the near-infrared photosensitizer that can be activated by the micro-acidic environment of a tumor provided by the present invention.

FIG2 is a synthetic route diagram of the activatable near-infrared photosensitizers LET-H, LET-Cl, LET-Br, and LET-I (collectively referred to as LET-R) of Examples 1 to 4 of the present invention.

FIG3 is a synthetic route diagram of the activatable near-infrared photosensitizer LET-BCy of Example 5 of the present invention.

FIG4 is a synthetic route diagram of the activatable near-infrared photosensitizer LET-Hcy-N of Example 6 of the present invention.

FIG5 is a synthetic route diagram of the activatable near-infrared photosensitizers LET-BHcy-ROS and LET-BHcy-GSH of Examples 7 to 8 of the present invention.

FIG. 6 is a pH-responsive absorption spectrum of the activatable near-infrared photosensitizers of Examples 1 to 8 of the present invention.

Figure 7 is a fluorescence emission spectrum diagram of the present invention, wherein a and b are pH-responsive fluorescence spectra of LET-H, with excitation wavelengths of 420 and 760 nm, respectively; c and d are pH-responsive fluorescence spectra of LET-I, with excitation wavelengths of 420 and 760 nm, respectively; and e is a pH-responsive fluorescence spectrum diagram of LET-Hcy-N, with an excitation wavelength of 660 nm.

In Figure 8, LET-R' (R = H, Cl, Br, I) represents the molecule after activation of the photosensitizer LET-R, a is a comparison of the degradation rates of DPBF by LET-R and LET-R' under 808 nm laser (power 0.2 W cm ^-2 ), b is a comparison of the singlet oxygen quantum yields of LET-R', and c is an electron paramagnetic resonance spectrum detecting the production of singlet oxygen by LET-H' or LET-I' with or without light.

FIG9 is a graph showing the test results of the cell killing ability of near-infrared photosensitizers LET-H and LET-I that can be activated under different illumination times.

In Figure 10, a is the fluorescence imaging of folic acid-modified nanoengineered LET-I (denoted as LET-I-FA) in mouse tumors and legs, b is the corresponding change in fluorescence intensity over time, c is the photoacoustic imaging of LET-I-FA in mouse tumors and legs, and d is the corresponding change in photoacoustic signal intensity over time.

In Figure 11, a is an evaluation diagram of the therapeutic effect of the photosensitizer LET-I-FA on the 4T1 tumor-bearing mouse model; b is a statistical diagram of the tumor weight of mice after the end of treatment; c is a diagram of the weight changes of mice in each treatment group during the treatment period, where LET-H-FA represents the folic acid-modified nanoengineered photosensitizer LET-H.

In FIG. 12 , a is a histological section diagram of the main organs of each group after treatment, and b is a diagram of the blood biochemical evaluation results of each group.

DETAILED DESCRIPTION

The present invention provides a near-infrared photosensitizer that can be activated by a microacidic environment of a tumor, and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The following describes it in detail through specific embodiments.

Example 1

The synthesis route for preparing the near-infrared photosensitizer LET-H that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG2 , and the specific synthesis steps are as follows:

Under nitrogen atmosphere, phosphorus oxychloride (8.7 mL, 58 mmol) was slowly added to a solution of 10 mL dichloromethane and 10 mL N, N-dimethylformamide. After stirring in an ice bath for 30 min, compound A1 (2.5 g, 25 mmol) was added. Stir at 80 °C for 6 h, and after cooling to room temperature, ice water was added and stirred overnight. The mixture was filtered, and the precipitate was washed with a small amount of ice water, then with ice ethanol, and finally dried in an oven to obtain a yellow solid compound B1 (4.1 g, yield 96%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ10.85 (s, 1H), 2.36 (m, 4H), 1.59 (m, 2H).

Compound D1 (1.07 g, 4.71 mmol) was added dropwise to a solution of compound C1 (500 mg, 3.14 mmol) in acetonitrile and refluxed for 1 d. After cooling, the mixture was poured into a mixture of 22.5 mL of dichloromethane and 2.5 mL of methanol. 10 mL of cold ether was added to the mixture, and the precipitated crystals formed were collected, washed with 3×3 mL of ether, and vacuum dried to obtain a light brown solid compound E1 (1.055 g, yield 87%). ¹ H NMR (400 MHz, CDCl ₃ ) δ7.73 (m, 1H), 7.59 (m, 3H), 4.89 (t, J＝8 Hz, 2H), 4.23 (t, J＝4 Hz, 2H), 3.17 (s, 3H), 2.38 (m, 2H), 2.00 (s, 3H), 1.67 (s, 6H). HRMS(ESI)calcd forC ₁₆ H ₂₂ NO ₂ ⁺ 260.1645,found 260.1636.

Under nitrogen protection, compound B1 (107 mg, 0.65 mmol), compound E1 (500 mg, 1.3 mmol) and sodium acetate (106 mg, 1.3 mmol) were added to 5 mL of acetic anhydride. The solution was heated to 70°C for 2 h, and the mixture was poured into cold ether, and the precipitate was collected to obtain a solid compound F1 (491 mg, yield 96%) with a metallic green luster. ¹ H NMR (500MHz, DMSO-d ₆ ) δ 8.29 (d, J = 10Hz, 2H), 7.66 (d, J = 10Hz, 2H), 7.45 (m, 4H), 7.30 (m, 2H), 6.36 (d, J = 15Hz, 2H), 4.30 (t, J = 7Hz, 4H), 4.09 (t, J = 6Hz, 4 H),2.72(t,J＝5Hz,4H),2.08(m,4H),1.94(s,6H),1.86(m,2H),1.69(s,12H).HRMS(ESI)calcd for C ₄₀ H ₄₈ ClN ₂ O ₄ ⁺ 655.3297,found 655.3275.

Under nitrogen protection, compound F1 (464 mg, 0.59 mmol) was dissolved in 30 mL of methanol. Potassium carbonate solid (164 mg, 1.19 mmol) was then added. The mixture was stirred at room temperature for 3 h, and the final product was synthesized after ring closure of intermediate compound G1. The solvent was removed by rotary evaporation, and 10 mL of dichloromethane was added. The resulting mixture was washed twice with 10 mL of saturated sodium bicarbonate aqueous solution, dried with anhydrous sodium sulfate, and then the solvent was removed by rotary evaporation. The crude product was purified by an aluminum oxide column (dichloromethane as eluent) to obtain a light yellow solid compound (208 mg, yield 62%), which is a near-infrared photosensitizer that can be activated by the slightly acidic environment of tumors, and is recorded as LET-H. ¹ HNMR (400MHz, DMSO-d ₆ ) δ 8.27 (d, J = 8Hz, 2H), 7.64 (d, J = 4Hz, 2H), 7.45 (m, 4H), 7.29 (td, J ₁ = 4.6Hz, J ₂ = 1.2Hz, 2H), 6.44 (d, J = 8Hz, 2H), 4.87 (bro, 2H), 4. 26(t,J＝4Hz,4H),3.50(bro,4H),2.70(t,J＝4Hz,4H),1.90(m,4H),1.68(m,14H). ¹³ C NMR (100MHz, CDCl ₃ )δ172.6,144.7,142.1,139.2,128.9,127.2,125.3,122.1,119.2,110.8,108.7,68.2,65.7,62.3,58.0,41.2,40.0,29.9,29.6,28.1,26.2,22.0 ,20.6,18.3.HRMS(ESI)calcd forC ₃₆ H ₄₃ ClN ₂ O ₂ ⁺ 570.3013,found[M+H] ⁺ 571.3078.

Example 2

The synthesis route for preparing the near-infrared photosensitizer LET-Cl that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG2 , and the specific synthesis steps are as follows:

A brown solid compound E2 was synthesized from compound C2 and D1 by the same synthesis steps as in Example 1 with a yield of 78%. ¹ H NMR (500 MHz, CDCl ₃ ) δ7.88 (d, J=5 Hz, 1H), 7.52 (s, 1H), 7.51 (d, J=2 Hz, 1H), 4.81 (t, J=5 Hz, 2H), 4.20 (t, J=5 Hz, 2H), 3.11 (s, 3H), 2.35 (m, 2H), 1.97 (s, 3H), 1.65 (s, 6H).

Compound E2 was used to obtain a green solid compound F2 with a yield of 92% by the same synthesis steps as in Example 1. ¹ HNMR (500 MHz, CDCl ₃ ) δ8.33 (d, J=10 Hz, 2H), 7.37 (dd, J ₁ =7 Hz, J ₂ =2 Hz, 2H), 7.33 (d, J=2 Hz, 2H), 7.12 (d, J=7 Hz, 2H), 6.34 (d, J=10 Hz, 2H), 4.38 (t, J=6 Hz, 4H), 4.18 (t, J=5 Hz, 4H), 2.79 (t, J=5 Hz, 4H), 2.21 (m, 4H), 2.04 (s, 6H), 1.98 (m, 2H), 1.73 (s, 12H).

Compound F2 was synthesized in the same manner as in Example 1 through a ring-closing reaction of intermediate compound G2 to obtain a yellow solid compound with a yield of 58%, which was a near-infrared photosensitizer activated by the slightly acidic environment of tumors and was recorded as LET-Cl. ¹ H NMR (500MHz, CD ₃ OD) δ8.45 (d, J = 15Hz, 2H), 7.59 (d, J = 5Hz, 2H), 7.44 (dd, J ₁ = 10Hz, J ₂ = 5Hz, 2H), 7.36 (d, J = 10Hz, 2H), 6.45 (d, J = 15Hz, 2H), 4.25 (t, J = 1 0Hz, 4H), 3.65 (t, J = 5Hz, 4H), 2.74 (t, J = 5Hz, 4H), 2.03 (m, 4H), 1.95 (m, 2H), 1.74 (s, 12H). ¹³ C NMR (100MHz, CD ₃ OD)δ174.1,145.6,144.5,142.5,132.1,129.9,128.9,124.1,113.5,59.3,50.7,42.5,31.0,28.2,27.3,22.1.HRMS(ESI)calcd for C ₃₆ H ₄₁ Cl ₃ N ₂ O ₂ ⁺ 64 0.2204,found[M+H] ⁺ 641.2263.

Example 3

The synthesis route for preparing the near-infrared photosensitizer LET-Br that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG2 , and the specific synthesis steps are as follows:

Compound C3 and D1 were used to synthesize light yellow solid compound E3 in the same manner as in Example 1 to prepare E1, with a yield of 82%. ¹ H NMR (500 MHz, CDCl ₃ ) δ7.80 (d, J=5 Hz, 1H), 7.71 (d, J=5 Hz, 1H), 7.68 (s, 1H), 4.85 (t, 2H), 4.22 (t, 2H), 3.12 (s, 3H), 2.36 (t, 2H), 2.00 (s, 3H), 1.67 (s, 6H).

Green compound F3 was obtained by the same procedure as in Example 1 using compound E3 in a yield of 95%. ¹ H NMR (500 MHz, CDCl ₃ ) δ8.34 (d, J=15 Hz, 2H), 7.52 (dd, J ₁ =7 Hz, J ₂ =1.5 Hz, 2H), 7.47 (d, J=1.5 Hz, 2H), 7.07 (d, J=5 Hz, 2H), 6.34 (d, J=10 Hz, 2H), 4.37 (t, J=5.5 Hz, 4H), 4.18 (t, J=4.5 Hz, 4H), 2.79 (t, J=5 Hz, 4H), 2.22 (m, 4H), 2.04 (s, 6H), 1.97 (m, 2H), 1.73 (s, 12H).

Compound F3 was used through the same synthesis steps as in Example 1 to obtain a yellow solid compound through ring closure of intermediate compound G3 with a yield of 67%, which is a near-infrared photosensitizer activated by the slightly acidic environment of tumors and is denoted as LET-Br. ¹ H NMR (500MHz, CD ₃ OD) δ8.45 (d, J = 15Hz, 2H), 7.72 (d, J = 2.5Hz, 2H), 7.58 (dd, J ₁ = 11Hz, J ₂ = 2.5Hz, 2H), 7.31 (d, J = 10.5Hz, 2H), 6.46 (d, J = 8Hz, 2H), 4.25 ( t,J＝9Hz,4H),3.65(t,J＝6.5Hz,4H),2.74(t,J＝7Hz,4H),2.02(m,4H),1.95(m,2H),1.74(s,12H). ¹³ C NMR (100MHz, CD ₃ OD)δ172.6,143.4,141.5,131.5,127.5,125.6,118.0,112.5,101.6,57.8,49.3,41.0,29.6,26.8,25.9,20.7.HRMS(ESI)calcd for C ₃₆ H ₄₁ Br ₂ ClN ₂ O ₂ ⁺ 728.1203,found[M+H] ⁺ 729.1257.

Example 4

The synthesis route for preparing the near-infrared photosensitizer LET-I that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG2 , and the specific synthesis steps are as follows:

A brown solid compound E4 was synthesized by similar steps using compound C4 and D1 with a yield of 60%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.32 (s, 1H), 8.03 (d, J=8 Hz, 1H), 7.78 (d, J=8 Hz, 1H), 4.51 (t, J=8 Hz, 2H), 4.13 (t, J=4 Hz, 2H), 2.81 (s, 3H), 2.18 (m, 2H), 1.93 (s, 3H), 1.54 (s, 6H). HRMS (ESI) calcd for C ₁₆ H ₂₂ INO ₂ ⁺ 386.0611, found 386.0608.

The green compound F4 was obtained by similar synthetic steps using compound E4 with a yield of 41%. ¹ H NMR (400 MHz, CDCl ₃ ) δ8.34 (d, J=11.2 Hz, 2H), 7.71 (dd, J ₁ =6.4 Hz, J ₂ =0.8 Hz, 2H), 7.65 (d, J=0.8 Hz, 2H), 6.97 (d, J=6.8 Hz, 2H), 6.36 (d, J=11.2 Hz, 2H), 4.37 (t, J=5.6 Hz, 4H), 4.18 (t, J=4.8 Hz, 4H), 2.79 (t, J=4 Hz, 4H), 2.21 (m, 4H), 2.04 (s, 6H), 1.72 (s, 14H). HRMS (ESI) calcd for C ₄₀ H ₄₆ ClI ₂ N ₂ O ₄ ⁺ 907.1230, found 907.1226.

Compound F4 was used through similar synthetic steps to obtain a yellow solid compound through intermediate compound G4 with a yield of 50%, which is a near-infrared photosensitizer activated by the slightly acidic environment of tumors and is recorded as LET-I. ¹ H NMR (500 MHz, CD ₃ OD) δ8.52 (d, J = 20 Hz, 1H), 8.40 (d, J = 15 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.86 (d, J = 2 Hz, 1H), 7.79 (dd, J ₁ = 10.5 Hz, J ₂ = 2 Hz, 1H), 7.74 (dd, J _{1 = 10.5 Hz, J 2} = 2 Hz, _1H ). =2Hz,1H),7.25(d,J＝10Hz,1H),7.11(d,J＝10Hz,1H),6.55(d,J＝20Hz,1H),6.26(d,J＝15Hz,1H),4.31(m,2H),4.24(m,2H),4.17(t,J＝5Hz,2H),3.66(m, 4H),2.77(m,4H),2.21(m,2H),1.97(m,2H),1.75(s,6H),1.73(s,6H). ¹³ C NMR (100MHz, CDCl ₃ )δ173.9,169.9,167.1,149.9,146.7,142.8,141.6,141.4,140.5,140.1,137.2,136.5,130.4,128.7,126.8,113.3,110.2,104.7,97.7,89.9,85 .9,60.3,56.9,47.3,41.6,39.8,27.3,27.1,26.9,25.5,25.1,19.9,19.7.HRMS(ESI)calcd for C ₃₆ H ₄₁ I ₂ ClN ₂ O ₂ ⁺ 822.0946,found[M+H] ⁺ 823.1015.

The structural formulas of the near-infrared photosensitizers LET-H, LET-Cl, LET-Br, and LET-I that can be activated by the slightly acidic environment of tumors prepared in the above Examples 1 to 4 are shown below:

Example 5

The synthesis route for preparing the near-infrared photosensitizer LET-BCy that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG3 , and the specific synthesis steps are as follows:

This embodiment provides a near-infrared photosensitizer that can be activated by a slightly acidic environment of a tumor. The synthesis route is shown in FIG3 . The specific synthesis method comprises the following steps:

Compound A5 (2 g, 11.8 mmol) and phosphorus pentasulfide (2.25 g, 11.8 mmol) were dissolved in 30 mL of pyridine and heated to 115°C under nitrogen protection and refluxed overnight. After the reaction was completed, the mixture was cooled and added to 400 mL of water, and then cooled and allowed to stand at 4°C for 3 to 4 hours. The solid was separated by filtration and washed with water three times to obtain a crude yellow solid compound B5, which was dried in vacuo and directly entered into the next step of reaction (1.79 g, yield 82.1%).

Compound B5 (12.4 g, 66.9 mmol) and iodomethane (5.0 mL, 80 mmol) were dissolved in 20 mL of acetone and heated under reflux at 45°C for 30 min. The precipitated product was crystallized from methanol to obtain a golden solid compound C5. The product was unstable and was directly used in the next step without purification (crude yield 76%).

A mixture of crude product compound C5 (16.4 g, 0.05 mol), compound D5 (14.4 g, 0.1 mol) and sodium acetate (8.2 g, 0.1 mol) in anhydrous ethanol was reacted at 50°C for 1 h. After the reaction was completed, the residue was cooled and filtered to separate, and washed with water and ethanol (volume ratio 1:1) to obtain compound E5 (13.8 g, yield 94%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.78 (s, 1H), 9.41 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 8 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 7.0 Hz, 1H), 7.69 (t, J = 8 Hz, 1H), 1.73 (s, 6H).

Compound E5 (443 mg, 1.5 mmol) and potassium carbonate (622 mg, 4.5 mmol) were mixed in 10 mL of N,N-dimethylformamide, stirred at 25° C. for 15 min, and then compound D1 (1.02 g, 4.5 mmol) was added dropwise. The mixture was then heated to 100° C., reacted for 12 h, then quenched with 250 mL of ice water, and extracted with dichloromethane. The crude product was purified by silica gel column chromatography (dichloromethane/methanol as eluent) to obtain solid compound F5 (367 mg, yield 62%). ¹ H NMR (500MHz, CDCl ₃ ) δ8.72(d,J＝7.0Hz,1H),8.58(d,J＝7.5Hz,1H),8.48(d,J＝7.0Hz,1H),8.27(d,J＝8.0Hz,1H),8.08(t,J＝7.5Hz,1H),7.95(t,J＝8Hz,1H ),5.06(s,2H),3.84(s,2H),3.53(s,3H),2.44(s,2H),1.26(s,6H).

Compound F5 (1.14 g, 2.9 mmol) was dissolved in 4 mL of acetic acid and the mixture was refluxed at 90 ° C for 1 h. 4 mL of concentrated hydrochloric acid was added dropwise to the refluxed mixture until the color changed from red to green. The mixture was cooled to room temperature and then saturated potassium iodide solution was added until the product began to precipitate. The product was filtered off, washed with ether, and vacuum dried to obtain solid compound G5 (314.7 mg, yield 48%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ9.0(d,J＝7.5Hz,1H),8.81(d,J＝8.0Hz,1H),8.52(d,J＝7.0Hz,1H),8.46(d,J＝8.0Hz,1H),8.150(t,J＝7.5Hz,1H),8.02(t,J＝8Hz, 1H), 4.75 (t, J = 7.0Hz, 2H), 3.55 (t, J = 5.5Hz, 2H), 3.26 (s, 3H), 2.13 (m, 2H).

Compound G5 (762.64 mg, 2.19 mmol), compound B1 (173 mg, 1 mmol) and sodium acetate (160 mg, 2 mmol) were dissolved in 5 mL of acetic anhydride and stirred at 40°C for 2 h to synthesize the condensation intermediate compound H5, followed by adding potassium carbonate solid (164 mg, 1.19 mmol) and stirring for 2 h, and then precipitating with 60 mL of ether to obtain a crude product, which was finally purified by column chromatography to obtain a solid compound, which is a near-infrared photosensitizer activated by a slightly acidic environment of a tumor, and is recorded as LET-BCy. HRMS (ESI) calcd for C ₃₈ H ₃₆ ClN ₂ O ₂ ⁺ 587.2460, found [M+H] ⁺ 587.2459.

The structural formula of the near-infrared photosensitizer LET-BCy activated by the slightly acidic environment of tumors prepared in Example 5 is as follows:

Example 6

The synthesis route for preparing the near-infrared photosensitizer LET-Hcy-N that can be activated by the slightly acidic environment of tumors in this embodiment is shown in FIG4 , and the specific synthesis steps are as follows:

Under nitrogen protection and ice bath conditions, phosphorus oxychloride (7.7mL, 82mmol) was slowly added dropwise to anhydrous N,N-dimethylformamide (7.9mL, 64mmol) and stirred for 15min, then compound A1 (5g, 50.9mmol) was added dropwise to the above reaction solution and stirred at room temperature for 2h. After the reaction was completed, the reaction solution was poured into 200g of ice water, sodium bicarbonate was slowly added, the pH value was adjusted to about 7, and then extracted with ethyl acetate several times, the organic phases were combined, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain orange oily compound A6 (6.8g, yield 93%). No further purification was required and it was directly used in the next step.

Compound A6 (0.97 g, 4 mmol), compound B6 (0.39 g, 2 mmol) and cesium carbonate (1.95 g, 6 mmol) were dissolved in 20 mL of anhydrous N,N-dimethylformamide and stirred at room temperature for 3 days. After the reaction was completed, the mixture was washed with dichloromethane and filtered, the filtrate was collected, washed with saturated brine for several times, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was then separated and purified by silica gel chromatography (dichloromethane) to obtain a yellow solid compound C6 (0.36 g, yield 64%). ¹ H NMR (400MHz, CDCl ₃ ) δ10.27 (s, 1H), 7.08 (m, 2H), 6.65 (s, 1H), 6.44 (s, 1H), 3.40 (d, J = 6.7Hz, 4H), 2.59 (m, 2H), 2.45 (t, J = 6.0Hz, 2H), 1.42 (s, 2H), 1.2 1(s,6H).

Under nitrogen protection, compound C6 (0.28 g, 1.0 mmol), compound E1 (0.32 g, 1.2 mmol), potassium carbonate (0.28 g, 2.0 mmol) and 5 mL of anhydrous acetic anhydride were added to a reaction flask and stirred at room temperature for 1 day. After the reaction, dichloromethane was added to the reaction solution, which was washed with water three times. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was then separated and purified by silica gel chromatography (dichloromethane/methanol, volume ratio 20:1) to obtain a dark green solid compound D6 (0.35 g, yield 67%). ¹ H NMR (500 MHz, CDCl ₃ )δ8.50(d,J＝14.1Hz,1H),7.56(s,1H),7.44(dd,J＝26.1,9.9Hz,3H),7.20(d,J＝8.0Hz,1H),6.82(d,J＝9.0Hz,1H),6.52(s,1H),6.18(d,J＝14.1Hz,1H),4 .35(t,J＝6.9Hz,2H),4.21(t,J＝5.9Hz,2H),3.56(q,J＝7.1Hz,4H),3.49(s,1H),2.83(m,4H),2.29(m,2H),1.95(s,3H),1.79(s,6H),1.72(s,2H),1.3 1(t,J=7.1Hz,6H).

Potassium carbonate (56 mg, 0.4 mmol) was added to a methanol (2 mL) solution of compound D6 (105 mg, 0.2 mmol) and stirred at room temperature overnight. D6 further underwent a ring-closing reaction via the intermediate compound E6. After the reaction was completed, the mixture was extracted with dichloromethane/saturated sodium bicarbonate solution, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was then separated and purified by an alkaline alumina chromatography column (petroleum ether/dichloromethane, volume ratio 50:1) to obtain a yellow solid, which was a near-infrared photosensitizer that can be activated by the ^slightly acidic environment of the tumor, recorded as LET-Hcy-N (86 mg, yield 89%). NMR(500MHz,MeOD)δ8.65(d,J＝12.6Hz,1H),7.55(d,J＝8.0Hz,2H),7.48(m,2H),7.39(d,J＝7.9Hz,1H),7.31(td,J＝7.4,1.0Hz,1H),6.93(dd,J＝9.0,2.4Hz,1H) ,6.73(d,J＝2.5Hz,1H),6.38 (d,J＝12.7Hz,1H),4.29(t,J＝7.2Hz,2H),3.69(t,J＝5.7Hz,2H),3.60(q,J＝7.1Hz,4H),2.76(dd,J＝26.1,6.3Hz,4H),2.08(m,2H),1.94(t,J＝5.2Hz,2H), 1.80 (s, 6H), 1.28 (d, J = 7.1Hz, 6H). HRMS(ESI)calcd for C ₃₂ H ₃₈ N ₂ O ₂ ⁺ 483.293, found[M+H] ⁺ 483.765.

The structural formula of the near-infrared photosensitizer LET-Hcy-N activated by the slightly acidic environment of tumors prepared in this example is as follows:

Example 7

The synthesis route of preparing the near-infrared photosensitizer LET-BHcy-ROS that can be activated by the micro-acid environment of tumors in this embodiment is shown in FIG5 , and the specific synthesis steps are as follows:

Compound A6 (1 g, 6.94 mmol) and compound A7 (0.88 g, 5.78 mmol) and cesium carbonate (5.65 g, 17.35 mmol) were dissolved in 40 mL of N,N-dimethylformamide and stirred at room temperature for 3 d. After the reaction was completed, the mixture was filtered and the filtrate was concentrated under vacuum. The residue was dissolved in 50 mL of dichloromethane and washed with distilled water (25×3 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. After chromatography purification (n-hexane/ethyl acetate, volume ratio 1:3), a yellow solid compound B7 (1.09 g, 78%) was obtained. The next step reaction was carried out directly.

Compound B7 (1 g, 2.19 mmol) was dissolved in anhydrous dichloromethane, and boron tribromide (2.2 mL, 22.6 mmol) was added dropwise in an ice bath at 0°C and nitrogen atmosphere. The reaction was stirred overnight at room temperature, then quenched with water and extracted with dichloromethane. The organic phase was rinsed with water three times, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was separated and purified by silica gel chromatography (dichloromethane/methanol, volume ratio 20:1) to obtain brown solid compound C7 (0.34 g, yield 34%). ¹ H NMR (500MHz, DMSO-d ₆ ): δ10.20 (s, 2H), 7.19 (d, J = 8.5Hz, 1H), 6.93 (s, 1H), 6.63 (d, J = 2.0Hz, 1H), 6.60 (dd, J = 8.5, 2.5Hz, 1H), 2.55 (m, 2H), 2.29 (t, J = 6Hz ,2H),1.64(m,2H).

Compound D7 (297 mg, 1.0 mmol) and potassium carbonate (138.21 mg, 1.0 mmol) were added to a dichloromethane solution of compound C7 (114.1 mg, 0.5 mmol), and the reaction solution was stirred at room temperature overnight. After the reaction was completed, the excess potassium carbonate was filtered off, the filtrate was dried over anhydrous sodium sulfate and concentrated, and the crude product was collected by silica gel column chromatography (dichloromethane/methanol, volume ratio 30:1) and the solvent was removed to obtain a yellow solid compound E7 (193 mg, yield 53%). HRMS (ESI) calcd for C ₂₇ H ₃₀ BO ₅ ⁺ 445.2108, found [M+H] ⁺ 445.217. Under nitrogen protection, compound G5 (68.9 mg, 0.2 mmol) and compound E7 (50 mg, 0.1 mmol) were dissolved in 10 mL of anhydrous ethanol, heated to 80 ° C and stirred for 12 h. The condensation intermediate compound F7 was synthesized, and then potassium carbonate solid (16.5 mg, 0.12 mmol) was added and stirred for 2 h, and then 30 mL of ether was used to precipitate to obtain a crude product. After the reaction was completed, the solvent was removed by spin drying. The crude product was separated and purified by column chromatography (dichloromethane/methanol, volume ratio ²⁰ :1) and collected and then spin dried. The obtained solid compound (45 mg, yield 55.4%) was a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor, and was recorded as LET-BHcy-ROS. NMR(500MHz,MeOD)δ8.89(d,J＝14.5Hz,1H),8.52(d,J＝7.5Hz,1H),8.15(d,J＝4.0Hz,1H),7.96(t,J＝7.5Hz,1H),7.73(dd,J＝8.0,4.5Hz,3H),7.59(m,2H),7.41 (m,4H),7.16(d,J =4.0Hz,1H),7.02(dd,J＝8.5,2.0Hz,1H),6.76(d,J＝14.5Hz,1H),5.24(s,2H),4.42(t,J＝7.0Hz,2H),3.65(t,J＝6.0Hz,2H),2.70(m,4H),2.04(m,2H),1 .88(m,2H),1.33(s,12H).

Example 8

The synthesis route for preparing the near-infrared photosensitizer LET-BHcy-GSH that can be activated by the micro-acid environment of tumors in this embodiment is shown in FIG5 , and the specific synthesis steps are as follows:

Under nitrogen protection and 0°C ice bath conditions, compound D8 (0.66 g, 2.5 mmol) and triethylamine (0.26 mL, 3 mmol) were added to a solution of compound C7 (114.1 mg, 0.5 mmol) in anhydrous dichloromethane in sequence, and the reaction solution was stirred overnight at room temperature. After the reaction, the reaction solution was washed with saturated sodium chloride and distilled water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain yellow solid compound E8 (106 mg, yield 48%). HRMS (ESI) calcd for C ₂₀ H ₁₅ N ₂ SO ₉ ⁺ 459.0420, found [M+H] ⁺ 459.067.

Under nitrogen protection, compound G5 (66.8 mg, 0.2 mmol) and compound E8 (50 mg, 0.1 mmol) were dissolved in 10 mL of anhydrous ethanol, and the reaction solution was heated to 80°C and stirred overnight to synthesize the condensation intermediate compound F8. Potassium carbonate solid (16.5 mg, 0.12 mmol) was then added and stirred for 2 h, and then precipitated with 30 mL of ether to obtain a crude product. After the reaction was completed, the solvent was removed by spin drying. The crude product was separated and purified by column chromatography (dichloromethane/methanol, volume ratio 20:1), collected and spin dried, and the obtained solid compound (34 mg, yield 45.9%) was a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor, and was recorded as LET-BHcy-GSH ^. HNMR(500MHz,MeOD)δ9.15(d,J＝15Hz,1H),8.96(d,J＝5Hz,1H),8.74(d,J＝10Hz,1H),8.52(dd,J＝10,5Hz,1H),8.37(d,J＝5Hz,1H),8.00(m,2H),7.84(d,J＝10Hz, 1H),7.60(d ,J＝5Hz,1H),7.43(d,J＝5Hz,1H),7.37(s,1H),7.14(dd,J＝10,5Hz,1H),7.03(d,J＝15Hz,1H),4.60(t,J＝5Hz,2H),3.67(t,J＝5Hz,2H),2.82(m,4H),2.14( m,2H),1.97(m,2H).

The structural formulas of the near-infrared photosensitizers LET-BHcy-ROS and LET-BHcy-GSH activated by the micro-acid environment of tumors prepared in the above Examples 7 to 8 are shown below:

Performance Testing

(1) Spectral test

Each photosensitizer prepared in Examples 1 to 8 was dissolved in dimethyl sulfoxide to prepare a photosensitizer mother solution with a concentration of 10 mM, and stored at 4°C for subsequent use. All tests were performed in a PBS buffer (1×) system containing 1% dimethyl sulfoxide (DMSO). 2 μL of photosensitizer mother solution was added to 2 mL of PBS buffer with different pH values, and the absorption spectra and fluorescence spectra of each photosensitizer under different pH conditions were obtained by UV-visible spectrophotometer and fluorescence spectrophotometer.

The absorption test results of each photosensitizer are shown in FIG6. Since the activation mechanism of each photosensitizer molecule is the same (as shown in FIG1), which is the cleavage of the ortho-ether oxygen bond of indole, the trend of the change of its UV-visible absorption spectrum with the change of pH is similar, that is, as the pH of the solution decreases, the absorbance of the photosensitizer molecule in the near-infrared light region gradually increases, while the absorbance in the visible light region gradually decreases. The fluorescence emission of the photosensitizer is taken as an example of LET-H, LET-I and LET-Hcy-N, and its fluorescence spectra are shown in a, b, c, d and e in FIG7. In the visible light region, the fluorescence emission intensity of the photosensitizer molecules LET-H and LET-I decreases with the increase of pH, while in the near-infrared light region, the fluorescence emission intensity of LET-H, LET-I or LET-Hcy-N increases with the increase of pH, indicating that the photosensitizer prepared by the present invention can be activated by a slightly acidic environment, thereby generating near-infrared absorption and fluorescence emission.

(2) Singlet oxygen generation test

The photosensitizers LET-R (R = H, Cl, Br, I) prepared in Examples 1 to 4 were tested. 1,3-diphenylisobenzofuran (DPBF) was selected as a singlet oxygen scavenger to measure the ability of each photosensitizer molecule to generate singlet oxygen.

First, prepare a 1mM DPBF stock solution for use, then add an appropriate volume of the newly prepared DPBF solution to DMSO, adjust the absorbance of DPBF at 415nm to about 1.0, and then add a photosensitizer solution of appropriate concentration. Determine its UV-visible light absorption spectrum when irradiated with a near-infrared laser for different times, and evaluate the ability of each photosensitizer to produce singlet oxygen by detecting the change in the DPBF absorbance at 415nm.

The test results are shown in a of FIG8 . Compared with the unactivated photosensitizer molecule LET-R (R=H, Cl, Br, I), the activated photosensitizer LET-R' (R=H, Cl, Br, I) has the ability to generate singlet oxygen, indicating that the photosensitizers prepared in Examples 1 to 4 of the present invention can produce near-infrared photodynamic therapy effects after being activated. In addition, LET-I' has a more efficient singlet oxygen generation ability, indicating that the ability of the photosensitizer molecule to generate singlet oxygen can be improved by modifying the halogen atoms of the photosensitizer molecular skeleton.

The singlet oxygen yield of the photosensitizer LET-R (R = H, Cl, Br, I) was tested using indocyanine green (ICG) as a reference. First, an appropriate volume of freshly prepared DPBF solution was added to dichloromethane, the absorbance of DPBF at 415 nm was adjusted to be about 1.0, and then an appropriate concentration of the test solution (photosensitizer molecule or ICG) was added. Subsequently, near-infrared laser irradiation was used to test the changes in the UV spectrum at different time points, and the singlet oxygen quantum yield of the molecule was calculated using the following formula (1):

Φ _Δ ＝Φ _MB *(k _ps *F _MB )/(k _MB *F _ps ) Formula (1)

Φ _Δ is the singlet oxygen quantum yield of the molecule, Φ _ICG is the singlet oxygen quantum yield of ICG. ps represents the molecule to be measured, and k represents the slope of the absorbance change of DPBF at 415 nm over time. The correction factor F is calculated by the following formula (2):

F＝1-10 ^OD formula (2)

In formula (2), OD represents the absorbance of the solution at the laser wavelength.

The test results are shown in Figure 8b, which further quantitatively confirms that the photosensitizer LET-I’ has a higher singlet oxygen generation ability.

The generation and type of singlet oxygen were further detected by electron paramagnetic resonance (ESR). Taking photosensitizers LET-H' and LET-I' as examples, 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TEMP) was used as a singlet oxygen scavenger. First, two groups of photosensitizer molecular mother solutions were taken, diluted and added with TEMP, one group was not treated with other methods, and the other group was irradiated with a near-infrared laser. A blank control was taken, and each group of samples was tested.

The test results are shown in Figure 8c. Near-infrared light irradiation of the sample containing activated photosensitizer LET-H’ or LET-I’ can produce three paramagnetic peaks of equal height (1:1:1), indicating that both photosensitizers LET-H’ and LET-I’ produce singlet oxygen.

(3) Evaluation of therapeutic effects at the cellular level

The standard MTT method was used to evaluate the effect of photodynamic therapy of photosensitizer molecules on the survival rate of mouse breast cancer cells (4T1 cells).

Cell culture was carried out in a DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin-streptomycin mixture) at 37°C and 5% CO ₂ in a constant temperature incubator. When the cell density reached 80%, relevant experiments were performed. Taking photosensitizers LET-H and LET-I as an example, 4T1 cells were inoculated in a 96-well plate (100 μL, 5×10 ³ cells/well) and cultured for 24 hours. After that, the original culture medium was discarded and a culture medium with a concentration of 20 μM photosensitizer molecules was added. After incubation for 6 hours, the culture medium in each well was replaced with fresh culture medium, irradiated with an 808nm (0.2W/cm ² ) laser for different times (0, 5, 10 and 20min), and placed in a cell culture incubator for continued incubation. After incubation for 12 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the cell culture incubator for another 1 hour, and then the absorbance at 450nm was detected with an ELISA reader.

The test results are shown in FIG9 . After 20 minutes of laser irradiation, the cell survival rate of the LET-I group was less than 30%, and was significantly lower than that of the LET-H group after 20 minutes of irradiation, indicating that the photosensitizer LET-I exhibits stronger phototoxicity.

(4) Visualized evaluation of photodynamic therapy effects at the in vivo level

Construction of a mouse tumor model for visualized photodynamic therapy.

Female BALB/c nude mice (6 weeks old) were purchased to establish a mouse tumor model. 4T1 cells in the logarithmic growth phase were digested and resuspended in serum-free medium. Before inoculation, the cells were kept on ice. All mice were randomly divided into groups, 5 in each group, and 4T1 cells were inoculated in the axilla of the right hind limb of the mouse, with 100 μL subcutaneous injection at each inoculation site. When the tumor volume reached 80 mm ³ , the photosensitizer molecules were injected into the mouse by intratumoral injection or tail vein injection, and the changes in fluorescence signals and photoacoustic signals in the tumor area over time were detected using a small animal fluorescence imaging system (IVISSpectrum) and a small animal photoacoustic imaging system (VisualSonics Vevo LAZR system). During the imaging experiment, all mice were anesthetized with isoflurane (2% in oxygen). Taking LET-H and LET-I as examples, in order to enhance the biocompatibility of the photosensitizer molecules, the photosensitizer molecules were nanoengineered using amphiphilic polymers modified with folic acid, which were recorded as LET-H-FA and LET-I-FA, respectively.

As shown in a and b in Figure 10, after LET-I-FA was injected intratumorally, the fluorescence signal of the tumor gradually increased, while the fluorescence intensity remained basically unchanged after subcutaneous injection into normal tissue (leg muscle). 20 minutes after injection, the fluorescence intensity of the mouse tumor was significantly higher than that of normal tissue. At the same time, the PBS control group showed a low and unchanged fluorescence signal in both tumors and normal tissues, indicating that LET-I-FA can be specifically activated in tumors and produce a strong fluorescence signal. At the same time, as shown in c and d in Figure 10, after LET-I-FA was injected intratumorally or subcutaneously into tumors or normal tissues, the photoacoustic signal intensity in the tumor reached the maximum after 20 minutes, while the photoacoustic signal of normal tissue remained basically unchanged and was lower than the signal intensity of the tumor. It further shows that LET-I-FA can be specifically activated in tumors and produce strong photoacoustic signals.

The tumor-bearing mice were randomly divided into 7 groups, namely: (1) PBS group; (2) simple illumination group; (3) LET-I-FA group; (4) LET-H-FA group; (5) LET-I illumination group; (6) LET-H-FA illumination group; (7) LET-I-FA illumination group. The photosensitizer solution of each group was injected into the mice through the tail vein at a dose of 10 μmol/kg and PBS at a dose of 100 μL. 12 hours after the injection, the group that needed laser irradiation was irradiated with 808 laser at a power of 0.2W/ ^cm2 for 30 minutes each. Starting from the time of administration, the weight changes of mice in each group were observed every other day. As shown in Figure 11a, the results show that there was no significant change in the weight of mice during the treatment process, indicating that the photosensitizer molecules used have good biosafety.

After 14 days of treatment, mice in each group were euthanized to obtain in vitro tumors, which were weighed and photographed. As shown in b and c in Figure 11, the tumors in the LET-I-FA irradiation group were significantly inhibited, and the treatment effect of this group was significantly better than that of the LET-H-FA irradiation group.

In addition, after the mice were dissected, the main organs (heart, liver, spleen, lung, and kidney) were fixed with paraformaldehyde and then stained with hematoxylin-eosin (H&E) to obtain tissue section images. The results shown in a of Figure 12 indicate that there were no significant changes in the main organs after treatment with the photosensitizer.

Three groups of mice were selected, each with 3 mice. LET-H-FA or LET-I-FA photosensitizer solution was injected into the mice at a dose of 10 μmol/kg and PBS at a dose of 100 μL through tail vein injection. After 14 days, blood from each group of mice was collected through orbital bleeding, centrifuged, and the upper serum was collected. The biochemical indexes of each group of mice were analyzed to evaluate the effects on the liver and kidney functions of mice. The results are shown in Figure 12b. There was no obvious abnormality in the blood biochemistry of each group of mice, which further indicated that the photosensitizer molecules used had good biosafety.

In summary, the near-infrared photosensitizer activated by the micro-acid environment of the tumor provided by the present invention can realize efficient and accurate tumor photodynamic therapy guided by fluorescence imaging/photoacoustic imaging. By introducing pH-responsive groups, the near-infrared photosensitizer has better tumor selectivity, thereby effectively reducing the phototoxicity of the photosensitizer to normal tissues. At the same time, this tumor-specific activation property gives the photosensitizer molecule a higher imaging signal-to-noise ratio, which is conducive to accurate visualization of photodynamic therapy. Moreover, by simply regulating the type of halogen atom substituted in the molecular skeleton, the ability of the photosensitizer molecule to produce singlet oxygen can be improved, effectively inhibiting tumor growth, and providing new ideas for the development of more new near-infrared photosensitizers.

It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims (10)

1. A near-infrared photosensitizer that can be activated by a slightly acidic environment of a tumor, characterized in that it has a chemical structure shown in any one of formula (I) to formula (VI): Wherein, R ₁ is H, F, Cl, Br or I atom, R ₂ is -OH, -NH ₂ , A is a self-eliminating group, and B is a biomarker responsive group. 2. The tumor microacid environment-activatable near-infrared photosensitizer according to claim 1, characterized in that the self-eliminating group A is 3. The tumor microacid environment-activatable near-infrared photosensitizer according to claim 1, characterized in that the biomarker responsive group B is: 4. The tumor slightly acidic environment-activatable near-infrared photosensitizer according to claim 1, characterized in that the photosensitizer represented by formula (I) to formula (VI) has the corresponding structures represented by formula (I') to formula (VI') in sequence after being activated in a slightly acidic environment: 5. The tumor slightly acidic environment activatable near-infrared photosensitizer according to claim 1, characterized in that it has the following structural formula: 6. A method for preparing a near-infrared photosensitizer activated by a microacidic environment of a tumor according to any one of claims 1 to 5, characterized in that it comprises the steps of: S1, compound 1 shown in formula 1 and compound 4 shown in formula 4 undergo condensation reaction to obtain compound 6 shown in formula 6; or the compound 2 represented by formula 2 and the compound 4 represented by formula 4 undergo a condensation reaction to obtain the compound 7 represented by formula 7; or the compound 3 shown in formula 3 and the compound 4 shown in formula 4 undergo a condensation reaction to obtain the compound 8 shown in formula 8; or the compound 1 represented by formula 1 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 9 represented by formula 9; or the compound 2 represented by formula 2 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain a compound 10 represented by formula 10; or the compound 3 represented by formula 3 and the compound 5 represented by formula 5 undergo a condensation reaction to obtain the compound 11 represented by formula 11; S2 and compound 6 undergo ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor as shown in formula (I); Or compound 7 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (II); Or compound 8 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (III); Or compound 9 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (IV); Or compound 10 undergoes ester hydrolysis reaction and ring closure reaction in sequence under alkaline conditions, thereby obtaining a near-infrared photosensitizer that can be activated by the slightly acidic environment of a tumor as shown in formula (V); Or compound 11 undergoes a ring-closing reaction under alkaline conditions to obtain a near-infrared photosensitizer that can be activated by the slightly acidic environment of the tumor as shown in formula (VI); Wherein, the structural formulas of compounds 1 to 11 are shown in the following formulas 1 to 11: The temperature of the condensation reaction in step S1 is 50-100° C., and the time of the condensation reaction is 2-12 hours; The alkaline conditions required for the ring-closing reaction in step S2 are one or more of potassium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, sodium hydride, ammonia water and triethylamine; and/or, the temperature of the ring-closing reaction is 0 to 50° C., and the time of the ring-closing reaction is 2 to 12 hours. 7. The method for preparing a tumor microacid environment-activatable near-infrared photosensitizer according to claim 6, characterized in that the compound 1 in step S1 is prepared by a substitution reaction between compound 12 and compound 13; Wherein, the temperature of the substitution reaction is 50-150° C., and the time of the substitution reaction is 24-72 hours; and/or, the solvent used in the substitution reaction is one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene and o-dichlorobenzene; The structural formulas of compound 12 and compound 13 are shown below: 8. The method for preparing a near-infrared photosensitizer activated by a tumor microacid environment according to claim 6, characterized in that the compound 3 in step S1 is prepared by the following steps: Compound 14 undergoes a sulfurization reaction to obtain compound 15; The compound 15 undergoes iodination reaction to obtain compound 16; The compound 16 undergoes a substitution reaction with the compound 17 to obtain the compound 18; The compound 18 undergoes a substitution reaction with the compound 13 to obtain the compound 19; The compound 19 undergoes a hydrolysis reaction to obtain the compound 3; The structural formulas of compounds 13 to 19 are shown below: 9. Use of a near-infrared photosensitizer that can be activated by a microacidic environment of a tumor as claimed in any one of claims 1 to 5, characterized in that it is used for preparing a tumor diagnostic agent and/or a tumor therapeutic agent. 10. The use of a near-infrared photosensitizer that can be activated by a micro-acidic environment of a tumor according to claim 9, characterized in that the tumor diagnostic agent comprises a fluorescent imaging agent or a photoacoustic imaging agent; and/or the tumor therapeutic agent comprises a photodynamic therapy agent or an agent for the combination of photodynamic therapy and other treatment regimens.